Use of genipin-1-β-d-gentiobioside in preparing drugs

Abstract

The use of genipin-1-β-D-gentiobioside in preparing drugs, comprising the use in drugs for treating respiratory inflammatory injuries caused by infections of coronaviruses, respiratory syncytial viruses or mycoplasma pneumoniae. The genipin-1-β-D-gentiobioside has the characteristics of reducing the viral load, lung index and lung tissue inflammatory cytokines in lung tissue after infections of coronaviruses, respiratory syncytial viruses or mycoplasma pneumoniae, and obviously preventing death caused by coronavirus infections.

Description

Application of genipin-1-β-D-gentiobioside in preparation of medicines Technical Field

[0001] The present invention relates to an application of genipin-1-β-D gentiobioside, in particular to an application of genipin-1-β-D gentiobioside in preparing a medicine for treating coronavirus infection, respiratory syncytial virus infection or mycoplasma pneumoniae infection. Background Art

[0002] Coronaviruses are the largest known group of RNA viruses. They are enveloped viruses with positive-strand RNA genomes and have a wide range of natural hosts. They are important pathogens in humans and vertebrates, infecting the respiratory tract, gastrointestinal tract, liver, kidneys, and nervous system, causing life-threatening pneumonia and bronchitis. In coronaviruses (CoV), the envelope spike glycoprotein (S) is responsible for CoV entry and intercellular transmission. After entering cells through endosome engulfment, coronaviruses release viral RNA and nucleocapsids into the cytoplasm, where they replicate within the host, completing the viral replication cycle. Coronaviruses can cause acute respiratory infections in humans, particularly the novel coronavirus, which can cause acute pneumonia and, in severe cases, death.

[0003] Currently, the primary treatment for coronavirus infection is based on small molecule compounds that act on the viral proliferation cycle, such as Paxilovid, Monuprivir, Azvudine, and VV116. Due to their single target and limited efficacy, these drugs are ineffective against viral mutations and drug resistance, and are particularly ineffective against severe or fatal cases caused by factors such as cytokine storm, disseminated intravascular coagulation, and immune deficiency.

[0004] Respiratory syncytial virus (RSV) is a circular negative-sense single-stranded RNA virus of the family Parainfluenzaviridae. Human respiratory syncytial virus is a common highly contagious virus that causes acute lower respiratory tract infection in infants and is the cause of high mortality in the elderly and children. RSV ranges from mild respiratory infection to severe respiratory infection, including pneumonia and bronchitis, and can cause serious complications such as respiratory failure. In the prior art, for respiratory syncytial virus pneumonia, ribavirin can be used as a preferred anti-RSV virus drug, but because ribavirin has multiple side effects, it is rarely used clinically. Therefore, for the treatment of respiratory syncytial virus pneumonia, comprehensive treatment (anti-infection, glucocorticoids, bronchoscope, anticoagulation, etc.) is currently mainly adopted, and clinical practice still lacks effective drugs.

[0005] Mycoplasma pneumoniae (MP) is a major pathogen causing respiratory infections in children. Mycoplasma pneumoniae pneumonia (MPP), the disease caused by MP, is currently the most common form of community-acquired pneumonia (CAP) in children aged 5 years and older in my country. Previous data have shown that during epidemics, MPP accounts for 20% to 40% of CAP in the general population and up to 70% in closed populations. Previously, the clinical manifestations of MPP were generally considered mild or even self-limiting. However, in recent years, an increasing number of severe MPP (SMPP) cases have been reported.

[0006] In the prior art, macrolide antibiotics are the first choice for the treatment of Mycoplasma pneumonia, but drug resistance has continued to emerge in recent years, resulting in poor efficacy; new tetracycline antibiotics, such as doxycycline and minocycline, are alternative drugs for the treatment of MPP, and have a definite effect on drug-resistant MPP, but may cause yellowing of the patient's teeth and enamel hypoplasia, so the new tetracycline antibiotics are relatively limited in clinical application; quinolone antibiotics are alternative drugs for the treatment of MPP, and have a definite effect on macrolide-resistant MPP, and are used to treat suspected or confirmed MP-resistant MUMPP, RMMP, and SMPP, but due to the risk of quinolone antibiotics causing cartilage damage in young animals and tendon rupture in humans, their clinical application is limited. In summary, at present, for Mycoplasma pneumonia, comprehensive treatment (anti-infection, glucocorticoids, bronchoscopy, anticoagulation, etc.) is still the main treatment, and there is still a lack of effective drugs in the clinic.

[0007] Summary of the Invention

[0008] The present invention aims to provide a use of genipin-1-β-D gentiobioside in the preparation of a drug. The present invention finds that genipin-1-β-D gentiobioside has the characteristics of reducing the viral load, lung index, and inflammatory cytokines in lung tissue after coronavirus infection, respiratory syncytial virus infection, or Mycoplasma pneumoniae infection, and has a significant protective effect against death caused by coronavirus infection.

[0009] The technical solution of the present invention is the use of genipin-1-β-D gentiobioside in the preparation of a drug for treating respiratory tract damage caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0010] The aforementioned application of genipin-1-β-D gentiobioside in the preparation of medicines, and the application of genipin-1-β-D gentiobioside in the preparation of medicines for treating inflammatory damage of pneumonia caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0011] The aforementioned application of genipin-1-β-D gentiobioside in the preparation of medicines, and the application of genipin-1-β-D gentiobioside in the preparation of medicines with death protection effect for treating pneumonia caused by coronavirus infection.

[0012] In the use of the aforementioned genipin-1-β-D gentiobioside in the preparation of drugs, the coronavirus includes any one of the new coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0013] The aforementioned genipin-1-β-D gentiobioside is used in the preparation of a drug, wherein the administration route of the genipin-1-β-D gentiobioside includes any one of aerosol inhalation, oral administration, injection, sublingual administration, spray or anal administration; and the dosage form of the drug includes any one of an inhaler, oral administration, injection, spray, film, and suppository.

[0014] An atomized inhalation agent containing genipin-1-β-D-gentiobioside is used to achieve the above application. The atomized inhalation agent comprises the following raw materials: genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator accounting for 0-0.9% of the weight of genipin-1-β-D-gentiobioside, and a solvent.

[0015] In the aforementioned aerosol inhalation, the preparation method of the aerosol inhalation comprises the following steps:

[0016] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0017] (2) adding an osmotic pressure regulator to the first solution at a temperature of 20 to 80° C. and stirring the first solution to obtain a second solution;

[0018] (3) adding a pH regulator and genipin-1-β-D gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0019] (4) Adding solvent to the third solution to make up the total volume of the solution required for preparation, stirring evenly, and obtaining a nebulized inhalation preparation of genipin-1-β-D-gentiobioside.

[0020] An injection containing genipin-1-β-D-gentiobioside, which realizes the above application, comprises the following raw materials: genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonic, and an injection solvent.

[0021] In the aforementioned injection, the preparation method of the injection comprises the following steps:

[0022] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0023] (2) adding an osmotic pressure regulator to the first solution at a temperature of 20 to 80° C. and stirring the first solution to obtain a second solution;

[0024] (3) adding a pH regulator and genipin-1-β-D gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0025] (4) Adding solvent to the third solution to make up the total volume of the solution required for preparation, stirring evenly, and obtaining an injection of genipin-1-β-D-gentiobioside.

[0026] A method for preparing genipin-1-β-D-gentiobioside, used for extracting genipin-1-β-D-gentiobioside for use in the above application, comprises the following steps:

[0027] a. Take Gardenia herbs, extract with water, and concentrate the extract under reduced pressure to a crude drug content of 0.03 to 0.2 g / ml;

[0028] b. The extract was applied to a macroporous resin column, first eluted with 1 to 5 column volumes of deionized water, then eluted with 1 to 5 column volumes of ethanol at a concentration of 10 to 20%, the ethanol eluate was collected, ethanol was recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, ethanol was added to an alcohol concentration of 90% by volume, allowed to stand, a precipitate was precipitated, filtered, the supernatant of the alcohol precipitate was passed through a neutral alumina column, sequentially eluted with 1 to 8 column volumes of ethanol at a concentration of 50 to 90%, the ethanol eluate was collected at a concentration of 50 to 60%, ethanol was recovered under reduced pressure, and dried to obtain a crude product;

[0029] c. The crude product was purified 2 to 3 times by hot-melt recrystallization with ethanol. After the refined product was dried, the ethanol was removed to obtain high-purity genipin-1-β-D gentiobioside.

[0030] In the aforementioned method for preparing genipin-1-β-D-gentiobioside, in step a, the specific steps of water extraction are: crushing the Gardenia jasminoides medicinal material, adding 12 times, 10 times, and 10 times the amount of water, respectively, and decocting three times, each decoction time being 1 to 1.5 hours; in step b, the weight ratio of the wet volume of the macroporous resin to the Gardenia jasminoides medicinal material is 3:2 to 3 ml / g, and the weight ratio of neutral alumina to the Gardenia jasminoides medicinal material is 1:3 to 3.5.

[0031] In the aforementioned method for preparing genipin-1-β-D gentiobioside, step b is specifically as follows: the gardenia extract is loaded onto an NKA-9 macroporous resin column, first eluted with 2 column volumes of deionized water, the loaded solution and the water eluate are collected, combined and loaded onto an X-5 macroporous resin, first eluted with 1 column volume of deionized water, then eluted with 5 column volumes of 10% ethanol by volume, the ethanol eluate is collected, ethanol is recovered under reduced pressure, and the relative density is 1.08 to 1.15 at 60°C. Ethanol is added to an alcohol volume concentration of 90%, allowed to stand, a precipitate is precipitated, filtered, and the supernatant of the alcohol precipitate is passed through a neutral alumina column, sequentially eluted with 6 column volumes of 90% ethanol by volume and 4 column volumes of 60% ethanol by volume, the ethanol eluate with a volume concentration of 60% is collected, ethanol is recovered under reduced pressure, and dried to obtain a crude product.

[0032] In the aforementioned method for preparing genipin-1-β-D gentiobioside, in step c, the method for recrystallizing the crude product after thermal dissolution in ethanol is specifically as follows: adding 0.5 to 1 times of anhydrous ethanol to the crude product, heating under reflux to dissolve it, filtering it while hot, allowing it to precipitate, and filtering it to obtain a refined product.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention discovered through animal experiments that genipin-1-β-D gentiobioside has a significant protective effect on the death of mice caused by the new coronavirus SARS-CoV-2 infection, and has a high lung index inhibition rate in mice infected with coronavirus, respiratory syncytial virus and Mycoplasma pneumonia. It can reduce the viral load of mice infected with coronavirus, respiratory syncytial virus or Mycoplasma pneumonia, and reduce the content of inflammatory factors TNF-α, IL-6 and IL-10 in the lung tissue of mice infected with coronavirus, respiratory syncytial virus or Mycoplasma pneumonia.

[0035] Therefore, genipin-1-β-D gentiobioside has a definite and significant therapeutic effect on viral pneumonia caused by coronavirus, respiratory syncytial virus, or mycoplasma pneumoniae infection by reducing multiple inflammatory cytokines after coronavirus infection, respiratory syncytial virus infection, or mycoplasma pneumoniae infection, inhibiting the inflammatory cytokine storm, and significantly reducing mortality, lung index, and lung tissue viral load. This shows that genipin-1-β-D gentiobioside has an anti-coronavirus, respiratory syncytial virus, and mycoplasma pneumoniae infection effect, and has a certain broad spectrum. It can treat respiratory diseases, severe pneumonia, and other diseases caused by coronavirus, respiratory syncytial virus, and mycoplasma pneumoniae infection, and has a death-protective effect on inflammatory damage; it can be used to prepare drugs for treating respiratory diseases caused by coronavirus infection, respiratory syncytial virus infection, or mycoplasma pneumoniae infection, drugs for pneumonia inflammatory damage, and drugs with pneumonia death protection effects.

[0036] The administration routes of genipin-1-β-D gentiobioside include aerosol inhalation, oral administration, injection, sublingual administration, spray and rectal administration. Genipin-1-β-D gentiobioside can be made into a variety of preparations commonly used by medical students.

[0037] The present invention also provides a method for preparing genipin-1-β-D-gentiobioside. The crude product produced by this method is free of most pigments and iridoid impurities, and the genipin-1-β-D-gentiobioside content in the crude product is greater than 60%. This method can efficiently separate and obtain the active fraction that meets the requirements of a new drug, wherein the active fraction content reaches greater than 50% of the extract. The resulting finished extract has a genipin-1-β-D-gentiobioside purity of greater than 96%.

[0038] In addition, during the preparation process of the present invention, toxic and harmful reagents are avoided and reusable macroporous resins are selected for purification. Therefore, the process route of the method is green, safe, relatively low in cost, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a graph showing the viral titer detection in the lung tissue of mice infected with the new coronavirus. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0041] The present invention discovers the application of genipin-1-β-D gentiobioside in preparing medicines for treating coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0042] Application of genipin-1-β-D gentiobioside in the preparation of a medicine for treating respiratory tract damage caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0043] Application of genipin-1-β-D gentiobioside in the preparation of a medicine for treating pneumonia inflammatory damage caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0044] The invention discloses an application of genipin-1-β-D gentiobioside in the preparation of a medicine having a death-protecting effect for treating pneumonia caused by coronavirus, respiratory syncytial virus or mycoplasma pneumoniae infection.

[0045] Furthermore, genipin-1-β-D gentiobioside is used in the preparation of drugs for treating increased levels of inflammatory factors TNF-α, IL-6, and IL-10 in lung tissue cells caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

[0046] The coronavirus includes any one of the new coronavirus SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

[0047] The genipin-1-β-D-gentiobioside of the present invention can be extracted from the traditional Chinese medicine Gardenia jasminoides, or prepared by artificial synthesis.

[0048] The method for extracting genipin-1-β-D-gentiobioside from the traditional Chinese medicine Gardenia jasminoides can be performed by the following steps:

[0049] a. Extraction of herbs: Take Gardenia herbs, crush Gardenia herbs, add 12 times, 10 times, 10 times the amount of water and boil three times, each boiling time is 1 to 1.5 hours, and the extract is concentrated under reduced pressure to a crude drug content of 0.03 to 0.2 g / ml;

[0050] b. Preparation of crude product: The extract is subjected to a macroporous resin column, preferably NKA-9 and X-5, with a wet volume to Gardenia medicinal material weight ratio of 3:2-3 ml / g. First, the extract is eluted with 1-5 column volumes of deionized water, then eluted with 1-5 column volumes of ethanol at a volume concentration of 10-20%, the ethanol eluate is collected, the ethanol is recovered under reduced pressure, and the extract is concentrated to 0.1 times the volume of the crude drug, ethanol is added to a volume concentration of 90%, the extract is allowed to stand, a precipitate is precipitated, and the extract is filtered. The supernatant of the alcohol precipitation is passed through a neutral alumina column with a specification of 100-200 mesh and a weight ratio of 1:3-3.5 to Gardenia medicinal material, and the extract is eluted with 1-8 column volumes of ethanol at a volume concentration of 50-90%, the ethanol eluate at a volume concentration of 50-60%, the ethanol is recovered under reduced pressure, and the extract is dried to obtain the crude product.

[0051] c. Preparation of Genipin-1-β-D-gentiobioside: The crude product was purified two to three times by ethanol hot dissolution and recrystallization. The specific purification method is as follows: the crude product was added to 0.5 to 1 part anhydrous ethanol, heated under reflux to dissolve, filtered while hot, allowed to stand to precipitate, and filtered to obtain a purified product. After drying the purified product, the ethanol was removed to obtain high-purity genipin-1-β-D-gentiobioside.

[0052] The administration route of genipin-1-β-D gentiobioside includes any one of aerosol inhalation, oral administration, injection, sublingual administration, spray or anal administration.

[0053] The atomization inhalation and injection drug delivery methods adopted by the present invention target the respiratory tract and lungs and deliver the drug directly to the respiratory tract and lungs in a mist-like form. This method has the advantages of rapid onset of action, high local drug concentration in the inflammatory lesions of the respiratory tract and lungs, low dosage, convenient application, and few systemic adverse reactions. It can be used as an important treatment for respiratory diseases.

[0054] The present invention also provides a medicine for treating coronavirus infection, comprising genipin-1-β-D gentiobioside and pharmaceutically acceptable excipients.

[0055] The dosage form of the drug includes any one of inhalation, oral preparation, injection, spray, film, and suppository.

[0056] Administration via inhalation, injection, spray, suppository and membrane (non-oral membrane) can avoid the first-pass liver-intestinal effect and prevent the genipin-1-β-D gentiobioside containing glycosidic bonds in the structure from being degraded by gastric acid and intestinal bacteria and becoming ineffective.

[0057] A nebulizer inhaler containing genipin-1-β-D-gentiobioside for the above application comprises the following raw materials: 20-200 mg of genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator accounting for 0-0.9% of the weight of genipin-1-β-D-gentiobioside, and 1-5 ml of a solvent.

[0058] The preparation method of an aerosol inhalation preparation containing genipin-1-β-D gentiobioside comprises the following steps:

[0059] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0060] (2) adding an osmotic pressure regulator to the first solution at a temperature of 20 to 80° C. and stirring the first solution to obtain a second solution;

[0061] (3) adding a pH regulator and genipin-1-β-D gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0062] (4) Adding solvent to the third solution to make up the total volume of the prepared solution, stirring evenly, and filtering with a 0.22 μm filter membrane or filter element to obtain a nebulized inhalation preparation of genipin-1-β-D-gentiobioside.

[0063] An injection containing genipin-1-β-D-gentiobioside for the above application comprises the following raw materials: 20-200 mg of genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5-7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonicity, and 1-5 ml of an injection solvent.

[0064] The preparation method of the injection comprises the following steps:

[0065] (1) measuring 40% to 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0066] (2) adding an osmotic pressure regulator to the first solution at a temperature of 20 to 80° C. and stirring the first solution to obtain a second solution;

[0067] (3) adding a pH regulator and genipin-1-β-D gentiobioside to the second solution to adjust the pH value of the second solution to a target value to obtain a third solution;

[0068] (4) Adding solvent to the third solution to make up the total volume of the solution required for preparation, stirring evenly, and obtaining an injection of genipin-1-β-D-gentiobioside.

[0069] Extraction Example 1:

[0070] The preparation method of genipin-1-β-D-gentiobioside comprises the following steps:

[0071] a. Extraction of herbs: Take 2kg of Gardenia herbs, crush the Gardenia herbs, add 12 times, 10 times, 10 times the amount of water and boil three times, each boiling time is 1 hour, the combined extracts were filtered and concentrated under reduced pressure to give a crude drug content of 0.033g / ml;

[0072] b. Preparation of crude product: The Gardenia extract was loaded onto an NKA-9 macroporous resin column, first eluted with 2 column volumes of deionized water, the sample and the water eluate were collected, combined and loaded onto an X-5 macroporous resin, first eluted with 1 column volume of deionized water, then eluted with 5 column volumes of 10% ethanol by volume, the ethanol eluate was collected, ethanol was recovered under reduced pressure, concentrated to 200 ml, the relative density at 60 ° C was 1.15, ethanol was added to an alcohol concentration of 90% by volume, allowed to stand, a precipitate was precipitated, filtered, the supernatant of the alcohol precipitate was passed through a 100 mesh neutral alumina column, followed by 6 column volumes of 90% ethanol by volume and 4 column volumes of 60% ethanol by volume elution, the ethanol eluate was collected at a concentration of 60% by volume, ethanol was recovered under reduced pressure, and dried to give a crude product;

[0073] c. Preparation of Genipin-1-β-D-gentiobioside: The crude product was added to 1.2 L of anhydrous ethanol, heated under reflux to dissolve, filtered while hot, allowed to stand to precipitate, and filtered with suction to obtain Refined Product 1. Refined Product 1 was added to 1.4 L of anhydrous ethanol, heated under reflux to dissolve, filtered while hot, allowed to stand to precipitate, filtered with suction, and the ethanol removed to obtain Refined Product 2. Refined Product 2 was dried (60°C, 0.7 MPa) and the ethanol removed to obtain 11.7 g of high-purity Genipin-1-β-D-gentiobioside with a mass content of 96.2%.

[0074] Extraction Example 2

[0075] The preparation method of genipin-1-β-D-gentiobioside comprises the following steps:

[0076] a. Extraction of medicinal materials: Take 5kg of Gardenia medicinal materials, crush the Gardenia medicinal materials, add 12 times the amount of water for the first time, extract for 1.5h, add 10 times the amount of water for the second time, extract for 1h, add 10 times the amount of water for the third time, extract for 1h, combine and filter the extracts, and concentrate under reduced pressure to give a crude drug content of 0.2g / ml;

[0077] b. Preparation of crude product: The Gardenia extract was loaded onto an NKA-9 macroporous resin column and eluted with 3 column volumes of deionized water. The sample and the water eluate were collected and combined and loaded onto an X-5 macroporous resin. The sample was first eluted with 1 column volume of deionized water, then eluted with 3 column volumes of ethanol at a volume concentration of 20%, the ethanol eluate was collected, ethanol was recovered under reduced pressure, and concentrated to 1 L, the relative density at 60 ° C was 1.08, ethanol was added to an alcohol volume concentration of 90%, allowed to stand, a precipitate was precipitated, filtered, and the supernatant of the alcohol precipitate was passed through a 100 mesh neutral alumina column, followed by 6 column volumes of 90% ethanol and 8 column volumes of 50% ethanol at a volume concentration of 50%, the ethanol eluate was collected, ethanol was recovered under reduced pressure, and dried to obtain a crude product;

[0078] c. Preparation of genipin-1-β-D gentiobioside: The crude product was added to 2.5 L of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to stand to precipitate, and filtered with suction to obtain purified product-1; purified product-1 was added to 2.5 L of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to stand to precipitate, filtered with suction, and the ethanol was removed to obtain purified product-2; purified product-2 was added to 2.8 L of anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, allowed to stand to precipitate, filtered with suction, and the ethanol was removed to obtain purified product-3. Purified product-3 was dried (60°C, 0.7 MPa) and the ethanol was removed to obtain 20.7 g of high-purity genipin-1-β-D gentiobioside with a mass content of 96.7%.

[0079] Preparation Example 1: Inhalation Administration-Solution for Inhalation

[0080] A nebulized inhalation solution containing genipin-1-β-D gentiobioside, the prescription composition of which is: 20 mg of genipin-1-β-D gentiobioside, citric acid for adjusting the pH value to 4.5, sodium chloride for adjusting the osmotic pressure to isotonic, and solvent to 1 ml.

[0081] The preparation method of the atomized inhalation solution comprises the following steps:

[0082] (1) measuring 40% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0083] (2) Control the temperature of the first solution to 25°C, add sodium chloride and stir until dissolved to obtain the second solution

[0084] (3) adding citric acid to adjust the pH value of the solution to 4.5 to obtain a third solution;

[0085] (4) adding genipin-1-β-D gentiobioside to the third solution, stirring evenly, and adding an appropriate amount of citric acid to maintain the pH of the solution at 4.5 to obtain a fourth solution;

[0086] (5) adding solvent to the fourth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fifth solution;

[0087] (6) Filter the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0088] (7) Fill the sixth solution into a 1 ml ampoule or vial and seal it to obtain the nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0089] Preparation Example 2: Inhalation Administration-Solution for Inhalation

[0090] A nebulized inhalation solution containing genipin-1-β-D-gentiobioside has the following prescription compositions: 100 mg of genipin-1-β-D-gentiobioside, 0.1% of citric acid, sodium citrate for adjusting pH value to 5.0, and solvent to 3 ml.

[0091] The preparation method of the above-mentioned atomized inhalation solution comprises the following steps:

[0092] (1) measuring 60% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0093] (2) While controlling the temperature of the first solution to be 50° C., adding citric acid to the first solution and stirring uniformly to obtain a second solution;

[0094] (3) adding sodium citrate to adjust the pH value of the solution to 5.0 to obtain a third solution;

[0095] (4) Genipin-1-β-D-gentiobioside was added to the second solution, stirred evenly, and an appropriate amount of sodium citrate was added to maintain the pH value of the solution at 5.0 to obtain a fourth solution;

[0096] (5) adding solvent to the fourth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fifth solution;

[0097] (6) Filter the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0098] (7) Fill the sixth solution into a 3 ml ampoule or vial and seal it to obtain the nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0099] Preparation Example 3: Inhalation Administration-Solution for Inhalation

[0100] A nebulized inhalation solution containing genipin-1-β-D gentiobioside has a prescription composition of: 200 mg of genipin-1-β-D gentiobioside, 0.5% of sodium citrate, sodium chloride for adjusting the osmotic pressure to isotonic, sodium hydroxide for adjusting the pH value to 7.0, and solvent to 5 ml.

[0101] The preparation method of the above-mentioned atomized inhalation solution comprises the following steps:

[0102] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0103] (2) Controlling the temperature of the first solution to 80° C., adding sodium citrate to the first solution and stirring uniformly to obtain a second solution;

[0104] (3) adding sodium chloride and stirring until dissolved to obtain a third solution;

[0105] (4) adding sodium hydroxide to adjust the pH value of the solution to 7.0 to obtain a fourth solution;

[0106] (5) adding genipin-1-β-D gentiobioside to the fourth solution, stirring evenly, and adding an appropriate amount of sodium hydroxide to maintain the pH value of the solution at 7.0 to obtain a fifth solution;

[0107] (6) adding solvent to the fifth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a sixth solution;

[0108] (7) Filtering the sixth solution through a 0.22 μm filter membrane or filter cartridge to obtain a seventh solution;

[0109] (8) The seventh solution is filled into a 5 ml ampoule or vial and sealed to obtain a nebulized inhalation solution of genipin-1-β-D-gentiobioside.

[0110] Preparation Examples 1-3, when in use, place the nebulizer (the atomization principle can be air compression, vibrating mesh or ultrasound) on a flat surface, and keep the machine as far away from textiles as possible when working to avoid textile lint clogging the machine's air inlet; after correctly installing the nebulizer cup according to the instructions. Open the medicine box, take out the ampoule or syringe, draw the liquid medicine with a syringe and transfer it to the nebulizer cup, sit or stand in an upright position to ensure normal breathing, confirm that the nebulizer mask covers the mouth and nose or the nebulizer mouthpiece is placed in the mouth, turn on the nebulizer button, start atomization, and continue inhaling until no more droplets are sprayed. In order to reduce the risk of infection, disease or contamination, clean and disinfect the nebulizer according to the instructions after the treatment.

[0111] After stability testing, the quality and atomization characteristics of the three formulation examples met the requirements after being placed at 25°C for 6 months.

[0112] Preparation Example 4: Inhalation Administration - Lyophilized Powder for Inhalation

[0113] A lyophilized powder for inhalation containing genipin-1-β-D-gentiobioside, the prescription composition of which is: 20 mg of genipin-1-β-D-gentiobioside, hydrochloric acid for adjusting the pH value to 4.5, and solvent to 1 ml.

[0114] The preparation method of the above-mentioned freeze-dried powder for inhalation is as follows:

[0115] (1) measuring 70% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0116] (2) controlling the temperature of the first solution to be 30° C., adding sodium hydroxide to the first solution, adjusting the pH value to 4.5, and stirring uniformly to obtain a second solution;

[0117] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring uniformly to obtain a third solution;

[0118] (4) measuring the pH value. If the pH value is not between 4.4 and 4.6, an appropriate amount of sodium hydroxide is added to adjust the pH value of the solution to 4.5 to obtain a fourth solution;

[0119] (5) adding solvent to the fourth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fifth solution;

[0120] (6) Filter the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0121] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0122] (8) The above samples were transferred to a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 ° C for 6 hours, vacuumize and heat to -15 ° C, perform sublimation drying, maintain for 10 hours, heat to 25 ° C, perform analytical drying, maintain for 4 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the lid is rolled to obtain the freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0123] Formulation Example 5: Inhalation Administration - Lyophilized Powder for Inhalation

[0124] A freeze-dried powder for inhalation containing genipin-1-β-D-gentiobioside has a prescription composition of: 80 mg of genipin-1-β-D-gentiobioside, 0.1% of citric acid, an appropriate amount of sodium citrate to adjust the pH value to 5.0, and a solvent to 2 ml.

[0125] The preparation method of the above-mentioned freeze-dried powder for inhalation is as follows:

[0126] (1) measuring 50% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0127] (2) While controlling the temperature of the first solution to 40° C., adding citric acid and sodium citrate to the first solution, adjusting the pH value to 5.0, and stirring uniformly to obtain a second solution;

[0128] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring evenly to obtain a third solution;

[0129] (4) measuring the pH value. If the pH value is not between 4.9 and 5.1, an appropriate amount of sodium citrate is added again to adjust the pH value of the solution to 5.0 to obtain a fourth solution;

[0130] (5) adding solvent to the fourth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fifth solution;

[0131] (6) Filter the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0132] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0133] (8) The above samples were transferred into a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 ° C for 6 hours, vacuumize and heat to -15 ° C, perform sublimation drying, maintain for 15 hours, heat to 30 ° C, perform desorption drying, maintain for 6 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the lid is rolled to obtain the freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0134] Preparation Example 6: Inhalation Administration - Lyophilized Powder for Inhalation

[0135] A lyophilized powder for inhalation containing genipin-1-β-D-gentiobioside has a formulation comprising: 200 mg of genipin-1-β-D-gentiobioside, 0.1% of citric acid, disodium hydrogen phosphate for adjusting the pH value to 7.0, and a solvent to 5 ml.

[0136] The preparation method of the above-mentioned freeze-dried powder for inhalation is as follows:

[0137] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0138] (2) While controlling the temperature of the first solution to 80° C., adding citric acid and disodium hydrogen phosphate to the first solution, adjusting the pH value to 7.0, and stirring uniformly to obtain a second solution;

[0139] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring uniformly to obtain a third solution;

[0140] (4) measuring the pH value. If the pH value is not between 6.9 and 7.1, an appropriate amount of disodium hydrogen phosphate is added to adjust the pH value of the solution to 7.0 to obtain a fourth solution;

[0141] (5) adding solvent to the fourth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fifth solution;

[0142] (6) Filter the fifth solution through a 0.22 μm filter membrane or filter element to obtain a sixth solution;

[0143] (7) Fill the sixth solution into a 10 ml vial and half-stopper it.

[0144] (8) The above samples were transferred to a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 ° C for 6 hours, vacuumize and heat to -15 ° C, perform sublimation drying, maintain for 25 hours, heat to 20 ° C, perform analytical drying, maintain for 10 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D gentiobioside for inhalation.

[0145] When using the samples prepared in Example 4-6 of the preparation, place the nebulizer (the atomization principle can be air compression, vibrating mesh or ultrasound) on a flat surface. When the machine is working, keep it as far away from textiles as possible to avoid textile lint clogging the air inlet of the machine; after correctly installing the nebulizer cup according to the instructions for use. Open the drug box, take out the vial of syringe, draw 1 to 5 ml of water for injection with a syringe, inject it into the vial of syringe, shake it to dissolve all the lyophilized powder, draw the liquid medicine with a syringe and transfer it to the nebulizer cup, sit or stand in an upright position to ensure normal breathing, confirm that the nebulizer mask covers the mouth and nose or the nebulizer mouthpiece is placed in the mouth, turn on the nebulizer button, start nebulization, and continue to inhale until no more droplets are sprayed. In order to reduce the risk of infection, disease or contamination, clean and disinfect the nebulizer according to the instructions after the treatment.

[0146] After stability testing, the contents and related substances of the freeze-dried powders obtained from the three formulation examples were relatively stable, and the atomization characteristics met the requirements.

[0147] Preparation Example 7: Injection Administration - Lyophilized Powder

[0148] A lyophilized powder for injection containing genipin-1-β-D-gentiobioside has a prescription composition of: 100 mg of genipin-1-β-D-gentiobioside, sodium chloride for adjusting the osmotic pressure to isotonic, sulfuric acid / sodium hydroxide for adjusting the pH value to 6.0, and water for injection to 3 ml.

[0149] The preparation method of the lyophilized powder for injection comprises the following steps:

[0150] (1) measuring 40% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0151] (2) controlling the temperature of the first solution to be 20° C., adding sodium chloride to the first solution and stirring uniformly to obtain a second solution;

[0152] (3) adding sodium hydroxide to the second solution, adjusting the pH to 6.0, and stirring uniformly to obtain a third solution;

[0153] (4) adding genipin-1-β-D gentiobioside to the third solution and stirring evenly to obtain a fourth solution;

[0154] (5) If the pH value is not between 5.9 and 6.1, an appropriate amount of sulfuric acid or sodium hydroxide is added again to adjust the pH value of the solution to 6.0 to obtain a fifth solution;

[0155] (6) adding water for injection to the fifth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a sixth solution;

[0156] (7) Filtering the sixth solution through a 0.22 μm filter membrane or filter cartridge to obtain a seventh solution;

[0157] (8) Fill the seventh solution into a 10 ml vial and half-stopper it.

[0158] (9) The above samples were transferred to a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 °C for 6 hours, vacuumize and heat to -20 °C, perform sublimation drying, maintain for 14 hours, heat to -10 °C, continue sublimation drying, maintain for 12 hours, after sublimation drying is completed, heat to 20 °C, perform desorption drying, keep warm for 4 hours, after freeze-drying is completed, fully press the stopper, take out of the box, and roll the cap.

[0159] Preparation Example 8: Injection Administration-Lyophilized Powder

[0160] A lyophilized powder for injection containing genipin-1-β-D-gentiobioside has a prescription composition of: 200 mg of genipin-1-β-D-gentiobioside, sodium chloride for adjusting the osmotic pressure to isotonic, and water for injection to 5 ml.

[0161] The preparation method of the lyophilized powder for injection comprises the following steps:

[0162] (1) measuring 90% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0163] (2) controlling the temperature of the first solution to be 50° C., adding sodium chloride to the first solution and stirring uniformly to obtain a second solution;

[0164] (3) adding genipin-1-β-D-gentiobioside to the second solution and stirring uniformly to obtain a third solution;

[0165] (4) adding water for injection to the third solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a fourth solution;

[0166] (5) filtering the fourth solution through a 0.22 μm filter membrane or filter element to obtain a fifth solution;

[0167] (6) Fill the fifth solution into a 10 ml vial and half-stopper it.

[0168] (7) The above samples were transferred to a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 ° C for 6 hours, vacuumize and heat to -20 ° C, perform sublimation drying, maintain for 20 hours, heat to -10 ° C, continue sublimation drying, maintain for 16 hours, after sublimation drying is completed, heat to 30 ° C, perform desorption drying, keep warm for 8 hours, after the freeze-drying is completed, fully press the stopper, take out of the box, and roll the cover to obtain.

[0169] Preparation Example 9: Injection Administration - Lyophilized Powder

[0170] A lyophilized powder for injection containing genipin-1-β-D-gentiobioside has a prescription composition of: 30 mg of genipin-1-β-D-gentiobioside, appropriate amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate to adjust the pH value to 7.0, sodium chloride to adjust the osmotic pressure to isotonic, and water for injection to 5 ml.

[0171] The preparation method of the lyophilized powder for injection comprises the following steps:

[0172] (1) measuring 60% (v / v) of the total amount of water for injection required to prepare the solution to obtain a first solution;

[0173] (2) controlling the temperature of the first solution to be 80° C., adding sodium chloride to the first solution and stirring uniformly to obtain a second solution;

[0174] (3) adding sodium dihydrogen phosphate and disodium hydrogen phosphate to the second solution, adjusting the pH to 7.0, and stirring uniformly to obtain a third solution;

[0175] (4) adding genipin-1-β-D gentiobioside to the third solution and stirring evenly to obtain a fourth solution;

[0176] (5) Measure the pH value. If the pH value is not between 6.9 and 7.1, add an appropriate amount of sodium dihydrogen phosphate / sodium hydrogen phosphate to adjust the pH value of the solution to 7.0 to obtain the fifth solution;

[0177] (6) adding water for injection to the fifth solution to make up the total volume required for preparing the solution, and stirring evenly to obtain a sixth solution;

[0178] (7) Filtering the sixth solution through a 0.22 μm filter membrane or filter cartridge to obtain a seventh solution;

[0179] (8) Fill the seventh solution into a 10 ml vial and half-stopper it.

[0180] (9) The above samples were transferred to a freeze dryer and freeze-dried according to the set freeze-drying curve: pre-freeze at -45 ° C for 6 hours, vacuumize and heat to -20 ° C, perform sublimation drying, maintain for 18 hours, heat to -10 ° C, continue sublimation drying, maintain for 15 hours, after sublimation drying is completed, heat to 20-30 ° C, perform desorption drying, and keep warm for 7 hours. After the freeze-drying is completed, the stopper is fully pressed, the sample is taken out of the box, and the cap is rolled to obtain the freeze-dried powder of genipin-1-β-D gentiobioside for injection.

[0181] When using the lyophilized powder prepared in Preparation Examples 7-9, open the drug packaging box, take out the vial, use a syringe to draw 1-5 ml of water for injection, inject it into the vial, shake it to dissolve the lyophilized powder completely, and then use it by intramuscular injection, intravenous injection, etc., or mix it with other infusion solutions and then use it by infusion.

[0182] The stability test of the lyophilized powders prepared in Preparation Examples 7-9 showed that the contents and related substances of the lyophilized powders were relatively stable after being stored at 25°C for 6 months.

[0183] Pharmacological Experimental Example 1: Protective Effect of Intravenous Injection of Genipin-1-β-D Gentianobioside on Death in a Mouse Pneumonia Model Infected with Human Novel Coronavirus

[0184] 1. Test materials

[0185] 1.1 Test drug: Genipin-1-β-D-gentiobioside, Appearance: White powder, Solubility: Very soluble in water. Mice were administered at 37.5 mg / kg and 75 mg / kg via intraperitoneal injection once daily for 5 consecutive days.

[0186] 1.2 Experimental Animals: hACE2 transgenic C57BL / 6 mice, 6–7 weeks old, weighing 18–25 g, 32 in total, provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., license number SCXK(Su)2018-008.

[0187] 1.3 Experimental conditions: The experiment was carried out in the ABSL-3 laboratory of Guangzhou Institute of Respiratory Health.

[0188] 2 Test methods

[0189] 2.1 Grouping and administration: hACE2 transgenic C57BL / 6 mice were divided into 4 groups, namely blank control group, SARS-CoV-2 infection group, genipin-1-β-D gentiobioside (37.5 mg / kg) administration group, and genipin-1-β-D gentiobioside (75 mg / kg) administration group, with 8 mice in each group. Except for the normal group, which was given PBS by intranasal drops, the mice in other groups were infected with 104 PFU of SARS-CoV-2 virus by intranasal drops. Two hours after infection, the mice in the drug-treated group were intraperitoneally injected once a day for 5 consecutive days. The death of mice was recorded every day after infection, and the 5-day mortality rate was calculated. After the end of the experiment, lung tissue was dissected and homogenized to detect the virus titer.

[0190] 2.2 Detection of virus titer in mouse lung tissue homogenate:

[0191] After removing mouse lung tissue, place it in a culture dish, mince it, transfer it to a homogenizer tube, and dilute it with saline at a 1:10 (w / v) ratio at 8000 rpm / min for 10 minutes. Perform all operations in an ice bath. Transfer the homogenate to a 1.5 mL EP tube and centrifuge it at 10000 rpm for 10 minutes at 4°C. Aspirate the supernatant, aliquot, and store at -80°C until needed.

[0192] VERO E6 cells in good growth condition were seeded in 96-well plates at 1×104 cells / well and cultured for 24 hours. After the cells attached to the wall and grew into a complete monolayer, the supernatant was discarded and the cells were washed twice with PBS. The supernatant of the frozen lung homogenate was thawed and diluted 10-fold to 10 -1 ~10 -5 Five concentrations were added to a 96-well plate. 100 μL of lung homogenate supernatant was added to each well of the plate. Blank control wells were also added with cell culture medium. Four replicate wells were prepared for each concentration and cultured in an incubator. Observation was continued for four consecutive days, with daily observations for cytopathic effect (CPE). The number of wells with CPE at each concentration gradient was recorded, and the TCID50 value for VERO E6 cells was calculated.

[0193] 3 Test results

[0194] 3.1 Protective effect of genipin-1-β-D gentiobioside on the death of mice infected with pneumonia caused by the new coronavirus ARS-CoV-2

[0195] Table 1 Protective effect of genipin-1-β-D gentiobioside on the death of mice infected with the new coronavirus ARS-CoV-2

[0196] The results in Table 1 show that no mice in the blank control group died during the experiment; after SARS-CoV-2 infection, the mortality rate of mice in the model control group was 75%; and after intraperitoneal injection of 37.5 mg / kg and 75 mg / kg of genipin-1-β-D-gentiobioside once daily for 5 consecutive days, the mortality rate of mice after SARS-CoV-2 infection was significantly reduced, with mortality rates of 37.5% and 25%, respectively. This indicates that genipin-1-β-D-gentiobioside has a significant protective effect against the mortality of mice caused by SARS-CoV-2 infection, with a good dose-effect correlation.

[0197] 3.2 Effect on virus titer in lung tissue of mice infected with SARS-CoV-2

[0198] [Corrected 26.12.2023 according to Rule 91] As shown in Figure 1, the results showed that after viral infection, a large amount of virus replication was observed in the lung tissues of mice in the model control group; administration of 37.5 mg / kg and 75 mg / kg doses of genipin-1-β-D gentiobioside significantly reduced the viral titer in the lung tissues of mice, which was significantly different from that of the model control group (P<0.05, P<0.01), and had a good dose-effect correlation.

[0199] Pharmacological Experiment 2: Therapeutic Effect of Intravenous Injection of Genipin-1-β-D Gentianobioside on Pneumonia Model in Mice Infected with Human Coronavirus 229E and OC43

[0200] 1. Test materials

[0201] 1.1 Test Drug: Genipin-1-β-D-gentiobioside, Appearance: White powder, Solubility: Very soluble in water. Mice were administered at 150 mg / kg, 75 mg / kg, and 37.5 mg / kg via intravenous injection once daily for 4 consecutive days.

[0202] 1.2 Positive drug: Chloroquine phosphate tablets, Sichuan Shenghe Pharmaceutical Co., Ltd., batch number: 2002114, production date: February 26, 2020, expiration date: January 2022. Specification: 0.25g / tablet, dosage: 0.5g / 60kg / d, oral.

[0203] 1.3 Experimental animals

[0204] Table 2 Experimental animals

[0205] 1.4 Virus Strains and Cells: Human coronavirus 229E (HCoV-229E) was provided by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences; human coronavirus HCoV-OC43 was purchased from the ATCC (American Pathology Collection Center). It was passaged in our laboratory and stored at −80°C until use. Human embryonic lung fibroblast MRC-5 cells were purchased from the Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen until use.

[0206] 1.5 Test equipment

[0207] Table 3 Test instruments

[0208] 1.6 Test site: ABSL-2 laboratory of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences

[0209] 2 Test methods

[0210] 2.1 Drug dosage design and preparation

[0211] Test drug: The dosage for mice was 150 mg / kg / d, 75 mg / kg / d, and 37.5 mg / kg / d;

[0212] Corresponding solutions were prepared with physiological saline and administered to mice by intravenous injection at a rate of 2 ml / 10 g / d, once a day for 4 consecutive days.

[0213] Chloroquine phosphate tablets: The clinical dosage is: 0.5g / 60kg / d for humans, orally.

[0214] The mouse dose is: Convert the clinical dose to the mouse dose: 0.5g / 60kg / d×11=0.09g / kg / d

[0215] The dosage for mice is: 20 ml / kg / d, administered by gavage;

[0216] The preparation concentration is: 0.09g / kg / d ÷ 20ml / kg / d = 0.0045g / ml.

[0217] 2.2 Virus passage

[0218] Take a 25cm2 culture flask of MRC-5 cells that have grown into a monolayer, discard the culture medium, rinse the cell surface three times with cell maintenance medium, add 5ml of cell maintenance medium, and then add 200μl of HCoV-229E or OC43 virus solution. Incubate in a 37°C incubator with a volume concentration of 5% CO2 for 72-96h. Observe the cell pathological changes under an inverted microscope every day until 80% of the cells show obvious pathological changes (CPE). Then, place the cell culture flask in a -80°C low-temperature refrigerator for freezing. After the virus solution is repeatedly frozen and thawed three times, it is used for virus titer determination.

[0219] 2.3 Virus titer determination

[0220] Take a 96-well plate with MRC-5 cells grown into a monolayer, discard the culture medium, wash the cells three times with cell maintenance medium, and then add 10 times (10 -1 ~10 -8 ) Inoculate HCoV-229E or OC43 virus at different titers for a total of eight dilutions, 100 μl / well, with quadruplicate wells for each dilution. A normal cell control was also established. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 72–96 hours. Cytopathic effects were observed daily under an inverted microscope and recorded for each well. The 50% cytopathic concentration (TCID50) was calculated using the Reed-Muench method.

[0221] 2.4 Construction and drug administration of human coronavirus mouse pneumonia model

[0222] BALB / c mice were randomly divided into three dose groups according to body weight: normal control group, model control group, chloroquine phosphate control group, and genipin-1-β-D gentiobioside, with 10 mice in each group, half male and half female. Except for the normal control group, mice in other groups were lightly anesthetized with ether and infected with 100TCID50HCoV-229E or OC43 intranasally, 50μl / mouse, once every other day, for a total of 2 infections. On the day of the first infection, each drug-treated group began intravenous administration once a day for 4 consecutive days. After weighing the body weight on the 5th day, the lungs were dissected and weighed, and the lung index and inhibition rate of the mice were calculated. Lung index = [lung wet weight (g) / body weight (g)] × 100 Lung index inhibition rate = (lung index of model control group - lung index of drug-treated group) / (lung index of model control group - lung index of normal control group) × 100%

[0223] 3 Test results

[0224] Table 4 The therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with human coronavirus 229E Note: Compared with the normal control group ## P<0.01, #P<0.05; compared with the model control group ** P<0.01, *P<0.05.

[0225] The results in Table 4 show that after infection with human coronavirus 229E virus, the lung index of mice was significantly increased, with a significant difference compared to the normal control group (P < 0.01). Four days after intravenous administration of genipin-1-β-D gentiobioside starting on the day of infection, all three doses tested significantly reduced the lung index of mice infected with the 229E virus, with significant differences compared to the model control group (P < 0.01). The lung index inhibition rates were 99.06%, 71.04%, and 75.12%, respectively. The drug's efficacy is comparable to that of chloroquine phosphate.

[0226] Table 5 The therapeutic effect of intravenous injection of genipin-1-β-D gentiobioside on the pneumonia model of mice infected with human coronavirus OC43 Note: Compared with the normal control group ## P<0.01, # P<0.05; **P<0.01, *P<0.05 compared with the model control group.

[0227] The results in Table 5 show that after mice were infected with human coronavirus OC43, the lung index of mice increased significantly, which was significantly different from that of the normal control group (P<0.01); after 4 days of intravenous injection of genipin-1-β-D gentiobioside starting on the day of infection, the three doses tested could significantly reduce the lung index of mice infected with OC43 virus, which was significantly different from that of the model control group (P<0.05, P<0.01), and the lung index inhibition rates were 39.93%, 42.34%, and 41.59%, respectively; the efficacy was comparable to that of chloroquine phosphate.

[0228] Pharmacological Experiment 3: Therapeutic Effect of Genipin-1-β-D Gentianobioside Aerosol Inhalation on Pneumonia Model in Mice Infected with Human Coronavirus 229E and OC43

[0229] 1. Test materials

[0230] 1.1 Test drug: Genipin-1-β-D-gentiobioside, batch number: 20210106, properties: white powder, solubility: very soluble in water.

[0231] 1.2 Experimental Animals: 140 BALB / c mice, SPF grade, weighing 13-15 g, half male and half female. They were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0232] 1.3 Strains and Cells: Human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43) were purchased from the American College of Traumatology (ATCC) with a TCID50 of 10⁻¹⁴. They were passaged in our laboratory and stored at -80°C until use. Human embryonic lung fibroblasts (MRC-5) were purchased from the Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen until use.

[0233] 1.4 Test reagents:

[0234] Table 6 Test reagents

[0235] 1.5 Test equipment:

[0236] Table 7 Test Instruments

[0237] 1.6 Test site: ABSL-2 laboratory of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences

[0238] 2 Test methods

[0239] 2.1 Drug dosage design and preparation

[0240] Test drug:

[0241] 1. In the 75 mg / ml genipin-1-β-D-gentiobioside group, the median particle size was 2.02±0.06 μm; in the 37.5 mg / ml genipin-1-β-D-gentiobioside group, the median particle size was 2.12±0.08 μm; in the 18.755 mg / ml genipin-1-β-D-gentiobioside group, the median particle size was 2.01±0.09 μm; in the 9.375 mg / ml genipin-1-β-D-gentiobioside group, the median particle size was 2.07±0.06 μm; the nebulization time for each group was 25 min.

[0242] 2. Chloroquine phosphate tablets: clinical dose is 0.5g / 60kg / d, oral; mouse dose is: 0.5g / 60kg / d × 11 = 0.09g / kg / d; mouse dosage is 20ml / kg / d, gavage; preparation concentration is 0.09g / kg / d ÷ 20ml / kg / d = 0.0045g / ml.

[0243] 2.2 Grouping and Dosing

[0244] Seventy ICR mice were randomly divided into seven groups according to weight: a normal control group, a model control group, a chloroquine phosphate control group, and four dose groups of genipin-1-β-D-gentiobioside (75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml), with 10 mice in each group. Except for the normal control group, the remaining mice were lightly anesthetized with isoflurane and intranasally infected with 100 TCID50 of coronavirus fluid (229E strain or OC43 strain) at 50 μl per mouse, and reinfected every other day. Nebulized administration began on the day of the first infection at a flow rate of 7.5 L / min. Four doses of genipin-1-β-D-gentiobioside (75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml) were administered for 25 minutes each. The chloroquine phosphate control group was gavaged with 0.2 ml / 10 g body weight once daily for four consecutive days. The normal control group and the model control group were administered distilled water nebulized for 25 minutes under the same conditions. Autopsies were performed on the fifth day, and the following parameters were measured:

[0245] ① The mice were weighed and then dissected, the whole lungs were removed and weighed, and the lung index and inhibition rate were calculated;

[0246] ② The left lung lobe of the mice was taken for viral nucleic acid detection, and the right lung lobe was taken for inflammatory factor content detection: IL-6, IL-10, and TNF-α;

[0247] The results were statistically analyzed using the t-test for intergroup comparison. Lung index = [lung wet weight (g) / body weight (g)] × 100 Lung index inhibition rate = (lung index of model control group - lung index of drug-treated group) / (lung index of model control group - lung index of normal control group) × 100%

[0248] 2.3 Detection of viral load in lung tissue (RT-PCR method)

[0249] ① Nucleic acid lysis treatment

[0250] After the mouse was dissected, the lung tissue was divided and stored in a -80℃ freezer. The mouse lung tissue was taken out of the -80℃ freezer, placed in a clean mortar, poured with a small amount of liquid nitrogen and ground into powder using a pestle. The powder was collected in a 1.5ml centrifuge tube and 1ml TRIzol was immediately added. Reagent, gently tap the bottom of the tube to mix the sample as soon as possible until it is resuspended; place the centrifuge tube horizontally at room temperature and incubate for 20 minutes; centrifuge at 4°C, 12000rpm, for 10 minutes; transfer the clear supernatant to a new 1.5ml centrifuge tube; add 0.2ml chloroform, cover the tube tightly, shake the centrifuge tube vigorously for 15 seconds, and incubate at room temperature for 2-3 minutes until the liquid separates; centrifuge at 4°C, 12000rpm, for 15 minutes; carefully transfer the clear supernatant to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, mix well, and incubate at room temperature for 30 minutes; centrifuge at 4°C, 12000rpm, for 10 minutes; discard the supernatant and gently wash the precipitate with 1ml 75% ethanol (so that the white precipitate floats lightly); centrifuge at 4°C, 7500rpm, for 5 minutes; aspirate the supernatant and briefly dry the RNA precipitate for 5-10 minutes; dissolve the precipitate with 20μl DEPC water and store in a -80°C refrigerator.

[0251] ② Nucleic acid determination

[0252] Nucleic acid treatment of control substances: DEPC-H2O was used as negative control. Positive control substances were 10 7 , 10 6 , 10 5 , 10 4 The number of copies / ml was serially diluted to 4 concentrations.

[0253] Reagent preparation: Take n × 18 μl HCoV-229E nucleic acid fluorescence PCR detection mixture, n × 1 μl internal control, and n × 1 μl RT-PCR enzyme (n is the number of reaction tubes), shake and mix for a few seconds, and centrifuge at 3000 rpm for a few seconds.

[0254] Add sample: Take 20μl of the above mixture and place it in a PCR tube. Then add 5μl each of sample nucleic acid extract, DEPC-H2O and positive control into the PCR tube, cover the tube tightly, centrifuge for a few seconds to allow all the liquid to settle at the bottom, and immediately proceed with the PCR amplification reaction.

[0255] PCR amplification: The reaction tube was placed on a quantitative fluorescence PCR instrument, and the cycle parameters were set as follows: 45°C × 10 min; 95°C × 15 min; then 95°C × 15 sec → 60°C × 60 sec, 40 cycles; single-point fluorescence detection was at 60°C, and the reaction system was 25 μl.

[0256] Fluorescence channel detection selection: select FAM and HEX / VIC / JOE channels.

[0257] ③Calculation method: Draw a standard curve according to the ct value of the positive control at different concentrations, and calculate the viral nucleic acid concentration of the sample according to the ct value of the sample.

[0258] 3 Test results

[0259] Table 8 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of HCOV-229E infected mice Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the model control group ** P<0.01, * P<0.05.

[0260] The results in Table 8 show that after mice were infected with the human coronavirus 229E strain, the lung index of the model control group was significantly increased, with a significant difference compared to the normal control group (P<0.01). After 4 days of aerosol inhalation of genipin-1-β-D gentiobioside starting on the day of infection, the lung index was significantly reduced in the four dose groups tested (75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml), all with significant differences compared to the model control group (P<0.01). The lung index inhibition rates were 109.19%, 64.22%, 65.59%, and 65.98%, respectively. The drug's efficacy was superior to that of chloroquine phosphate.

[0261] Table 9 Effect of nebulized inhalation of genipin-1-β-D gentiobioside on the viral load in lung tissue of mice infected with HCOV-229E Note: Compared with the normal group ## p<0.01, compared with the model group ** p<0.01.

[0262] The results in Table 9 show that there is no viral nucleic acid expression in the lung tissue of normal mice. After the mice were infected with the human coronavirus 229E strain virus, the coronavirus nucleic acid was significantly expressed in the lung tissue of the mice. After 4 days of aerosol inhalation treatment with genipin-1-β-D gentiobioside starting on the day of infection, the expression of coronavirus 229E nucleic acid in the lung tissue of mice in the four dose groups of genipin-1-β-D gentiobioside at 75 mg / ml, 37.5 mg / ml, 18.75 mg / ml, and 9.375 mg / ml was significantly reduced, and there were significant differences compared with the model control group (P<0.01).

[0263] Table 10 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the pneumonia model of HCOV-OC43 infected mice Note: Compared with the normal control group ## P<0.01, # P<0.05; compared with the model control group ** P<0.01, * P<0.05.

[0264] The results in Table 10 show that after mice were infected with human coronavirus OC43 strain, the lung index of mice in the model control group was significantly increased, which was significantly different from that in the normal control group (P<0.01). After 4 days of aerosol inhalation treatment with genipin-1-β-D gentiobioside starting on the day of infection, the four dose groups of genipin-1-β-D gentiobioside tested, namely 75 mg / ml, 37.5 mg / ml, 18.75 mg / ml and 9.375 mg / ml, were able to significantly reduce the lung index of infected mice, which was significantly different from that in the model control group (P<0.05, P<0.01). The lung index inhibition rates were 102.88%, 90.30%, 75.73% and 49.42%, respectively.

[0265] Table 11 Effect of nebulized inhalation of genipin-1-β-D gentiobioside on the viral load in lung tissue of mice infected with HCOV-OC43 Note: Compared with the normal group ## p<0.01, compared with the model group ** p<0.01.

[0266] The results in Table 11 show that there is no viral nucleic acid expression in the lung tissue of normal mice. After mice were infected with human coronavirus OC43 strain virus, the coronavirus nucleic acid was significantly expressed in the lung tissue of mice in the model control group. After 4 days of aerosol inhalation treatment with genipin-1-β-D gentiobioside starting on the day of infection, the expression of coronavirus nucleic acid in the three dose groups of genipin-1-β-D gentiobioside at 75 mg / ml, 37.5 mg / ml, and 18.75 mg / ml was significantly reduced, and there were significant differences compared with the model control group (P<0.01).

[0267] Pharmacological Experiment Example 4: Effects of Genipin-1-β-D Gentianobioside on Inflammatory Cytokines in Lung Tissue of Mice Infected with HCOV-229E

[0268] Administer the sample via aerosol inhalation as described above. Weigh the lung tissue and add an appropriate amount of physiological saline to prepare a 10% tissue homogenate. Homogenize the tissue using a high-throughput tissue grinder and centrifuge in a low-temperature high-speed centrifuge at 3000 rpm for 10 minutes at 4°C. Aspirate the supernatant, aliquot, and store at -80°C until needed. Avoid repeated freeze-thaw cycles. For assay, measure absorbance at 450 nm using a microplate reader according to the kit instructions.

[0269] Table 12 Effects of genipin-1-β-D gentiobioside on cytokines in lung tissue of mice infected with human coronavirus 229E Compared with the normal control group ## P<0.01, # P<0.05; compared with the model group, ** P<0.01.

[0270] The results in Table 12 show that after the human coronavirus 229E infection model was established, the levels of TNF-α, IL-6, and IL-10 cytokines in the lung tissue of the mice in the model group were significantly increased, which was significantly different from the normal control group (P<0.01); nebulized inhalation of genipin-1-β-D gentiobioside was given for 4 days starting on the day of infection. All four dose groups could reduce the levels of TNF-α, IL-6, and IL-10 in the lung tissue of the mice, which was significantly different from the model group (P<0.01).

[0271] Pharmacological Example 5: Therapeutic Effect of Genipin-1-β-D Gentianobioside Aerosol Inhalation on Pneumonia Model in Mice Infected with Respiratory Syncytial Virus (RSV)

[0272] 1. Test materials

[0273] 1.1 Test drug

[0274] Genipin-1-β-D gentiobioside preparation, specifications: 5ml; 375mg, batch number: 230608; properties: light yellow liquid; storage: store at room temperature.

[0275] 1.2 Experimental animals

[0276] Balb / c mice, SPF grade, weighing 9-11 g, 50 mice, half male and half female, license number: SCXK (Beijing) 2016-0006.

[0277] 1.3 Virus strains

[0278] Human respiratory syncytial virus (RSV), VR1580TM, was purchased from ATCC, passaged in our laboratory, and stored in a -80°C refrigerator until use.

[0279] 1.4 Test instruments

[0280] Table 13 Test Instruments

[0281] 2 Test methods and results

[0282] 2.1 Effects on the lung index and lung index inhibition rate of mice

[0283] Sixty BALB / C mice, weighing 10±1g, were randomly divided into four groups: a normal control group, a model control group, a high-dose group (75mg / ml) of genipin-1-β-D-gentiobioside, and a low-dose group (37.5mg / ml) of genipin-1-β-D-gentiobioside, with 10 mice per group, half male and half female. Except for the normal control group, mice in all groups were lightly anesthetized with isoflurane and intranasally infected with respiratory syncytial virus (RSV) (35μl per mouse). On the day of infection, each group began nebulizing the drug for 25 minutes once daily for four consecutive days. The normal and model control groups received nebulized saline under the same conditions. On the fifth day, mice were weighed and sacrificed by cervical dislocation. Lung tissue was dissected and weighed, and the lung index and lung index inhibition rate were calculated. Lung tissue was retained for viral load, pathological analysis, and detection of inflammatory factors. The results were statistically analyzed using the t-test for intergroup comparisons. Lung index = [lung wet weight (g) / body weight (g)] × 100 Lung index inhibition rate = (lung index of model control group - lung index of drug-treated group) / (lung index of model control group - lung index of normal control group) × 100%

[0284] Table 14 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the RSV-infected mouse pneumonia model Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0285] The results in Table 14 show that the lung index of mice in the RSV model control group was significantly increased, which was significantly different from that in the normal control group (p<0.01). After the 75 mg / ml and 37.5 mg / ml doses of genipin-1-β-D gentiobioside were administered by aerosol for 25 minutes each time, once a day, for 4 consecutive days, the lung index of mice was significantly reduced, which was significantly different from that in the model control group (p<0.01). The lung index inhibition rates were 88.35% and 68.79%, respectively.

[0286] 2.2 Effects on viral load in mouse lung tissue

[0287] Nucleic acid detection in lung tissue (RT-PCR method)

[0288] ① Nucleic acid lysis treatment

[0289] After the mouse was dissected, the lung tissue was divided and stored in a -80℃ freezer. The mouse lung tissue was taken out of the -80℃ freezer, placed in a clean mortar, poured with a small amount of liquid nitrogen and ground into powder using a pestle. The powder was collected in a 1.5ml centrifuge tube and 1ml TRIzol was immediately added. Reagent, gently tap the bottom of the tube to mix the sample as soon as possible until it is resuspended; place the centrifuge tube horizontally at room temperature and incubate for 20 minutes; centrifuge at 4°C, 12000rpm, for 10 minutes; transfer the clear supernatant to a new 1.5ml centrifuge tube; add 0.2ml chloroform, cover the tube tightly, shake the centrifuge tube vigorously for 15 seconds, and incubate at room temperature for 2-3 minutes until the liquid separates; centrifuge at 4°C, 12000rpm, for 15 minutes; carefully transfer the clear supernatant to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, mix well, and incubate at room temperature for 30 minutes; centrifuge at 4°C, 12000rpm, for 10 minutes; discard the supernatant and gently wash the precipitate with 1ml 75% ethanol (so that the white precipitate floats lightly); centrifuge at 4°C, 7500rpm, for 5 minutes; aspirate the supernatant and briefly dry the RNA precipitate for 5-10 minutes; dissolve the precipitate with 20μl DEPC water and store in a -80°C refrigerator.

[0290] ② Nucleic acid determination

[0291] Nucleic acid treatment of control substances: DEPC-H2O was used as a negative control. The positive control substance was serially diluted to 4 concentrations, namely 107, 106, 105, and 104 copies / ml.

[0292] Reagent preparation: Take n×18 μl RSV nucleic acid fluorescence PCR detection mixture, n×1 μl internal control, and n×1 μl RT-PCR enzyme (n is the number of reaction tubes), shake to mix for a few seconds, and centrifuge at 3000 rpm for a few seconds.

[0293] Add sample: Take 20μl of the above mixture and place it in a PCR tube. Then add 5μl each of sample nucleic acid extract, DEPC-H2O and positive control into the PCR tube, cover the tube tightly, centrifuge for a few seconds to allow all the liquid to settle at the bottom, and immediately proceed with the PCR amplification reaction.

[0294] PCR amplification: The reaction tube was placed on a quantitative fluorescence PCR instrument, and the cycle parameters were set as follows: 45°C × 10 min; 95°C × 15 min; then 95°C × 15 sec → 60°C × 60 sec, 40 cycles; single-point fluorescence detection was at 60°C, and the reaction system was 25 μl.

[0295] Fluorescence channel detection selection: select FAM and HEX / VIC / JOE channels.

[0296] Note: When using an ABI series thermal cycler, be sure to select "none" for both the passive reference and quencher options.

[0297] Calculation method: Draw a standard curve according to the ct value of the positive control at different concentrations, and calculate the viral nucleic acid concentration of the sample according to the ct value of the sample.

[0298] Table 15 The therapeutic effect of nebulized inhalation of genipin-1-β-D gentiobioside on the RSV-infected mouse pneumonia model

[0299] The results in Table 15 show that the RSV viral load in the lung tissue of mice in the RSV model control group was significantly increased; after the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiobioside were administered by aerosol for 25 minutes each time, once a day, for 4 consecutive days, the RSV viral load in the lung tissue of mice was significantly reduced; the inhibition rates were 56.08% and 57.54%, respectively.

[0300] Pharmacological Example 6: Therapeutic Effect of Genipin-1-β-D Gentianobioside Atomized Inhalation on Mice Infected with Mycoplasma Pneumoniae

[0301] 1.1 Test drug: Genipin-1-β-D-gentiobioside preparation, specification: 5 ml; 375 mg, batch number: 230608.

[0302] 1.2 Positive drug: Azithromycin capsules: Batch number: 23032004, production date: 2023.03.14, expiration date: 2025.02. Produced by Sunflower Pharmaceutical Co., Ltd. Ingredients: The main ingredient of this product is azithromycin. Properties: This product is a capsule, and the contents are white or off-white crystalline powder. Indications: Pneumonia caused by Mycoplasma pneumoniae. Specifications: 0.25g / capsule. Usage and dosage: Oral. Adults: 0.5g once a day for 3 consecutive days. Children: 10mg / kg daily based on body weight. Storage conditions: Sealed and stored in a dry place.

[0303] 1.3 Experimental animals

[0304] 100 Balb / c mice, SPF grade, weighing 13-15 g, half male and half female.

[0305] 1.4 Virus strain: Mycoplasma pneumoniae (MP), VR15531 TM .

[0306] 1.5 Test equipment

[0307] Table 16 Test Instruments

[0308] 1.6 Test Reagents

[0309] Table 17 Test reagents

[0310] 2 Test methods and results

[0311] 2.1 Effects on the lung index and lung index inhibition rate of mice

[0312] Fifty BALB / C mice, weighing 14±1g, were randomly divided into five groups: a normal control group, a model control group, an azithromycin control group, a high-dose genipin-1-β-D-gentiobioside group (75mg / ml) nebulized for 15 minutes, and a low-dose genipin-1-β-D-gentiobioside group (37.5mg / ml) nebulized for 15 minutes. Each group consisted of 10 mice, half male and half female. Except for the normal control group, mice in each group were lightly anesthetized with isoflurane and intranasally infected with Mycoplasma pneumoniae (50μl per mouse) for three consecutive days. On the day of infection, each treatment group received nebulized saline once daily for four consecutive days. The normal and model control groups received nebulized saline under the same conditions. On the fifth day, mice were weighed and sacrificed by cervical dislocation. Lung tissue was then dissected and weighed, and the lung index and lung index inhibition rate were calculated. Inflammatory cytokines and serum CRP levels were measured, and lung tissue was obtained for pathological examination. The results were statistically analyzed using the t-test for intergroup comparison. Lung index = [lung wet weight (g) / body weight (g)] × 100 Lung index inhibition rate = (lung index of model control group - lung index of drug-treated group) / (lung index of model control group - lung index of normal control group) × 100%

[0313] Table 18 The therapeutic effect of genipin-1-β-D gentiobioside on the pneumonia model of MP-infected mice Note: Compared with the normal control group ## p<0.05; compared with the model control group ** p<0.01.

[0314] The results in Table 18 show that the lung index of the MP model control group was significantly increased, and there was a significant difference compared with the normal control group (p<0.01); after 4 days of aerosol administration of genipin-1-β-D gentiobioside, the lung index of mice in the high and low dose groups of genipin-1-β-D gentiobioside were significantly different from those in the model control group (p<0.01), and the results of the two batches of experiments had good repeatability.

[0315] 2.2 Detection of inflammatory factors in mouse lung tissue (ELISA method)

[0316] ① Sample Collection and Storage: Tissue Homogenate Samples: After weighing lung tissue from mice, collect lung tissue from 8 mice per group and store at -80°C. Homogenize the tissue using the MPFastprep-245G rapid sample preparation instrument and centrifuge at -4°C, 3000 rpm, for 10 minutes. Aspirate the supernatant, aliquot, and store at -80°C until needed. Avoid repeated freeze-thaw cycles.

[0317] ② Remove the microplate from the sealed bag, which has been equilibrated to room temperature. Add 50 μL of the standard sample of varying concentrations to the corresponding wells. Add 10 μL of the experimental sample to each well, followed by 40 μL of diluent. Add 100 μL of HRP to each well, except for the blank well. Seal the wells with adhesive tape and incubate at 37°C for 1 hour. Wash the plate with wash buffer four times. After the final wash, invert the plate and pat dry with absorbent paper to remove any residual liquid. Add 50 μL each of substrates A and B to each well. Seal the wells with adhesive tape. Incubate at 37°C for 15 minutes. Afterward, add 50 μL of stop solution to each well. Within 15 minutes, measure the absorbance at 450 nm using a microplate reader. Calculate the results.

[0318] Table 19 The therapeutic effect of genipin-1-β-D gentiobioside on the pneumonia model of MP-infected mice Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0319] The results in Table 19 show that the inflammatory factors IL-6, IL-1β, and TNF-α in the lung tissue of the MP model control group were significantly increased, which was significantly different from that of the normal control group (p<0.01); after 4 days of nebulization administration of high and low doses of genipin-1-β-D gentiobioside, the inflammatory factor content of pneumonia mice was significantly reduced, which was significantly different from that of the model control group (p<0.01).

[0320] 2.3 Detection of CRP in mouse serum (ELISA method)

[0321] ① Sample collection and storage: Blood was collected from the mouse orbit and allowed to stand for 2 hours. The sample was then centrifuged at 2000g for 10 minutes. The supernatant was aspirated and stored at -80℃ until use. Avoid repeated freezing and thawing. The sample was diluted 3000 times with normal saline for CRP detection.

[0322] ② Remove the microplate from the sealed bag, which has been equilibrated to room temperature. Add 100 μL of standard and sample concentrations to the corresponding wells, and incubate at room temperature for 2 hours. Wash the plate with washing solution, repeating this process five times. Add 100 μL of biotinylated antibody and incubate at room temperature for 60 minutes. Wash the plate five times, then add 100 μL of horseradish peroxidase and incubate in the dark for 20 minutes. After washing the plate five times, add the color developer and incubate for 20 minutes. Finally, add 50 μL of stop solution. Measure the absorbance at 450 nm using a microplate reader within 15 minutes. Calculate the results.

[0323] Table 20 The therapeutic effect of genipin-1-β-D gentiobioside on the pneumonia model of MP-infected mice Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0324] The results in Table 20 show that the serum CRP content of mice in the MP model control group was significantly increased, which was significantly different from that in the normal control group (p<0.01); after 4 days of nebulization administration of high and low doses of genipin-1-β-D gentiobioside, the CRP level of inflammatory factors in pneumonia mice was significantly reduced, which was significantly different from that in the model control group (p<0.01).

[0325] 2.4 Pathological changes of mouse lung tissue (HE staining)

[0326] Normal control group: The alveolar surface was smooth without obvious deformation or rupture; the bronchiolar epithelial cells were arranged neatly without edema, degeneration or necrosis; there was no fibrous tissue proliferation and inflammatory cell infiltration in the pulmonary interstitium; and the tissue structure was normal.

[0327] In the model control group, microscopy revealed extensive alveolar congestion, edema, and exudation, with partial alveolar wall rupture, forming alveolar fusion and consolidation, and mild or moderate thickening of the alveolar septa, accompanied by a large infiltration of inflammatory cells, primarily neutrophils, with a small number of lymphocytes, macrophages, and plasma cells. Bronchial mucosal epithelial cells showed slight or mild edema and degeneration, with a small amount of necrotic and desquamated epithelial cells and inflammatory cells visible in the bronchial lumen. These results were significantly different from those in the normal control group (P < 0.01).

[0328] In the BD-77 high-dose group, the mice had focal mild or mild congestion and edema of the alveoli, mild or mild thickening of the alveolar wall, accompanied by infiltration of a small amount of inflammatory cells, mainly neutrophils. Some animals had mild or mild edema of the bronchial epithelium, and the arrangement of bronchial epithelial cells was more regular, which was significantly alleviated compared with the model group (P < 0.1).

[0329] In the BD-77 low-dose group, microscopic examination revealed slight or mild thickening of the alveolar wall of the mice, accompanied by a small amount of inflammatory cell infiltration, which was significantly alleviated compared with the model group (P < 0.1); multifocal mild or mild congestion and edema of the alveoli were alleviated compared with the model group, but no statistical difference was observed; bronchial mucosal epithelial cells showed slight to moderate edema and degeneration, and some bronchial epithelial cells were necrotic, which was not significantly different from the model group.

[0330] Table 21 The therapeutic effect of BD-77 on MP-infected mouse pneumonia model

[0331] Pharmacological Experiment Example 7: Therapeutic Effect of Intravenous Injection of Genipin-1-β-D Gentianobioside on LPS-Induced Pneumonia Model in Mice

[0332] 1. Test materials

[0333] 1.1 Drug: Genipin-1-β-D-gentiobioside preparation, Specification: 5 ml; 375 mg, Batch No.: 230608; Properties: Light yellow liquid; Storage: Store at room temperature.

[0334] 1.2 Animals: 60 ICR mice, SPF grade, weighing 18-20 g, half male and half female, provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2021-0006; they were housed in an ABSL-2 biosafety room.

[0335] 1.3 Reagents and Instruments: Escherichia coli endotoxin (LPS) was produced by Sigma, lot number: 057M4013V. Isoflurane was produced by Jiangsu Hengfengqiang Biotechnology Co., Ltd., lot number: 20211202. Production date: 20211224, expiration date: 20231223. MC 1.8 biological safety cabinet was produced by Thermo Fisher Scientific. BSA3202S-CW and BSA323S-CW electronic balances were produced by Sartorius Instruments Co., Ltd. AL-204 METTLER TOLEDO electronic balance was produced by Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0336] 2. Test methods and results:

[0337] 2.1 Effects on the lung index and lung index inhibition rate of mice

[0338] Sixty healthy mice, weighing 18-20 g (half male and half female), were stratified by body weight and then randomly divided into a normal control group, a model control group, and three dose groups of genipin-1-β-D-gentiobioside (150 mg / kg / day, 75 mg / kg / day, and 37.5 mg / kg / day), along with a positive control group of sivelestat sodium (50 mg / kg / day). Each group consisted of 10 mice (half male and half female). Each treatment group received 20 ml / kg of LPS intravenously once daily for two consecutive days, while the normal and model control groups received saline. One hour after the second day of treatment, all mice, except the normal control group, were lightly anesthetized with isoflurane and intranasally administered 0.05 ml of 20 mg / ml LPS in saline solution to induce a pneumonia model. Six hours after infection, the animals were sacrificed and dissected. The lungs were removed and weighed, and the lung index was calculated (lung index = lung weight / body weight × 100%).

[0339] Table 22 Effects of genipin-1-β-D gentiobioside on LPS-induced pneumonia model in mice Note: Compared with the normal control group ## p<0.01; compared with the model control group ** p<0.01.

[0340] The results in Table 22 show that 6 hours after LPS inhalation, the lung index of mice in the model control group was significantly increased, with a significant difference compared to the normal control group (p<0.01); the lung index of mice in the high and medium dose groups of genipin-1-β-D gentiobioside was significantly reduced, with a significant difference compared to the model control group (p<0.01, p<0.05). This indicates that the 150 mg / kg and 75 mg / kg dose groups of genipin-1-β-D gentiobioside have a protective effect against LPS-induced pneumonia in mice.

[0341] 2.2 Detection of inflammatory factors in mouse lung tissue (ELISA method)

[0342] ① Sample Collection and Storage: Tissue Homogenate Samples: After weighing the lung tissue of mice, collect lung tissue from mice #1, 2, 3, 6, 7, and 8 in each group and store at -4°C. Homogenize the tissue using an ultrasonic cell disruptor and centrifuge in a low-temperature high-speed centrifuge at 1000 rpm for 10 minutes at -4°C. Aspirate the supernatant, aliquot, and store at -80°C until needed. Avoid repeated freezing and thawing.

[0343] ② Remove the microplate from the sealed bag that has been equilibrated to room temperature and add different concentrations of standards, experimental samples, or quality control products to the corresponding wells, 100μL per well. Seal the reaction wells with adhesive tape and incubate at room temperature for 2 hours. Wash the plate with wash solution and repeat this operation 4 times. After the last wash, invert the plate and pat dry any residual liquid on absorbent paper; add 100μL of enzyme-labeled detection antibody to each microwell. Seal the reaction wells with adhesive tape and incubate at room temperature for 2 hours. Repeat the plate wash operation in step 4, add 100μL of chromogenic substrate to each microwell, and incubate at room temperature for 30 minutes. Protect from light. Within 30 minutes after adding 100μL of stop solution to each microwell, measure the absorbance at 450nm using a microplate reader. Calculate the results.

[0344] Table 23 Effects of genipin-1-β-D gentiobioside on LPS-induced pneumonia model in mice Note: Compared with the normal control group ## p<0.01; compared with the model control group * p<0.05, ** p<0.01.

[0345] The results in Table 23 show that 6 hours after LPS inhalation, the levels of IL-6, IL-10, and TNF-α in the lung tissues of mice in the model control group were significantly increased, with significant differences compared to the normal control group (p<0.01, p<0.05); the levels of IL-6, IL-10, and TNF-α in the groups treated with genipin-1-β-D gentiobioside were significantly reduced, with significant differences compared to the model control group (p<0.01, p<0.05). This indicates that both the 150 mg / kg and 75 mg / kg doses of genipin-1-β-D gentiobioside have a protective effect against LPS-induced pneumonia in mice.

Claims

1. Application of genipin-1-β-D-gentiobioside in the preparation of a drug, characterized in that: Application of genipin-1-β-D-gentiobioside in the preparation of a drug for treating respiratory tract injury caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

2. The application of genipin-1-β-D-gentiobioside in the preparation of a drug according to claim 1, characterized in that: Application of genipin-1-β-D-gentiobioside in the preparation of a drug for treating pulmonary inflammatory injury caused by coronavirus, respiratory syncytial virus or Mycoplasma pneumoniae infection.

3. The application of genipin-1-β-D-gentiobioside in the preparation of a drug according to claim 3, characterized in that: Application of genipin-1-β-D-gentiobioside in the preparation of a drug with a death protection effect on pneumonia caused by coronavirus infection.

4. The application of genipin-1-β-D-gentiobioside in the preparation of a drug according to claim 1, characterized in that: The coronavirus includes any one of SARS-CoV-2, human coronavirus 229E, and human coronavirus OC43.

5. The application of genipin-1-β-D-gentiobioside in the preparation of a drug according to claim 1, characterized in that: The administration route of the genipin-1-β-D-gentiobioside includes any one of aerosol inhalation, oral administration, injection, sublingual administration, spraying or rectal administration; the dosage form of the drug includes any one of inhalants, oral preparations, injections, sprays, membranes, and suppositories.

6. An aerosol inhalant containing genipin-1-β-D-gentiobioside for realizing the application according to any one of claims 1-5, characterized in that: The aerosol inhalant includes the following raw materials: genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5 - 7.0, an osmotic pressure regulator accounting for 0 - 0.9% of the weight of genipin-1-β-D-gentiobioside, and a solvent.

7. The aerosol inhalant containing genipin-1-β-D-gentiobioside according to claim 6, characterized in that: The preparation method of the aerosol inhalant includes the following steps: (1) Measure 40% - 90% (v / v) of the total amount of the solution to be prepared of injection water to obtain a first solution; (2) Under the condition that the temperature of the first solution is 20 - 80 °C, add the osmotic pressure regulator to the first solution and stir evenly to obtain a second solution; (3) Add the pH regulator and genipin-1-β-D-gentiobioside to the second solution, and adjust the pH value of the second solution to the target value to obtain a third solution; (4) Add the solvent to the third solution to make up the volume to the total amount of the solution to be prepared, and stir evenly to obtain the aerosol inhalant of genipin-1-β-D-gentiobioside.

8. An injection containing genipin-1-β-D-gentiobioside for realizing the application according to any one of claims 1-5, characterized in that: The injection includes the following raw materials: genipin-1-β-D-gentiobioside, a pH regulator for adjusting the pH value to 4.5 - 7.0, an osmotic pressure regulator for adjusting the osmotic pressure to isotonicity, and an injection solvent.

9. The injection containing genipin-1-β-D-gentiobioside according to claim 8, characterized in that: The preparation method of the injection includes the following steps: (1) Measure 40% - 90% (v / v) of the total amount of the solution to be prepared of injection water to obtain a first solution; (2) Under the condition that the temperature of the first solution is 20 - 80 °C, add the osmotic pressure regulator to the first solution and stir evenly to obtain a second solution; (3) Add the pH regulator and genipin-1-β-D-gentiobioside to the second solution, and adjust the pH value of the second solution to the target value to obtain a third solution; (4) Add the solvent to the third solution to make up the volume to the total amount of the solution to be prepared, and stir evenly to obtain the injection of genipin-1-β-D-gentiobioside.

10. A preparation method of genipin-1-β-D-gentiobioside for extracting genipin-1-β-D-gentiobioside in the application according to any one of claims 1-5, characterized in that: It includes the following operation steps: a. Take Gardenia jasminoides Ellis herbs, extract with water, and concentrate the extract under reduced pressure so that the crude drug content in the extract is 0.03 - 0.2 g / ml; b. The extract is loaded onto a macroporous resin column, first eluted with 1 - 5 column volumes of deionized water, then eluted with 1 - 5 column volumes of ethanol with a volume concentration of 10 - 20%. The ethanol eluate is collected, ethanol is recovered under reduced pressure, concentrated to 0.1 times the volume of the crude drug, ethanol is added to make the ethanol volume concentration 90%, left to stand, precipitate is separated out, filtered. The supernatant of ethanol precipitation is passed through a neutral alumina column and eluted successively with 1 - 8 column volumes of ethanol with a volume concentration of 50 - 90%. The ethanol eluate with a volume concentration of 50 - 60% is collected, ethanol is recovered under reduced pressure, and dried to obtain the crude product; c. The crude product is refined 2 - 3 times by the method of hot dissolution and recrystallization with ethanol. After the refined product is dried, ethanol is removed to obtain high - purity genipin - 1 - β - D - gentiobioside.

11. The preparation method of genipin-1-β-D-gentiobioside according to claim 10, characterized in that: In step a, the specific steps of water extraction are as follows: The gardenia medicinal materials are crushed and decocted 3 times with 12 times, 10 times, and 10 times the amount of water respectively, and each decoction time is 1 - 1.5 hours; In step b, the ratio of the wet volume of the macroporous resin to the weight of the gardenia medicinal materials is 3:2 - 3 ml / g, and the ratio of neutral alumina to the weight of the gardenia medicinal materials is 1:3 - 3.

5.

12. The preparation method of genipin-1-β-D-gentiobioside according to claim 10, characterized in that: Specifically for step b, the gardenia extract is loaded onto an NKA - 9 macroporous resin column, first eluted with 2 column volumes of deionized water, the loading solution and the water eluate are collected and combined, then loaded onto an X - 5 macroporous resin, first eluted with 1 column volume of deionized water, then eluted with 5 column volumes of ethanol with a volume concentration of 10%. The ethanol eluate is collected, ethanol is recovered under reduced pressure, concentrated to a relative density of 1.08 - 1.15 at 60 °C, ethanol is added to make the ethanol volume concentration 90%, left to stand, precipitate is separated out, filtered. The supernatant of ethanol precipitation is passed through a neutral alumina column and eluted successively with 6 column volumes of ethanol with a volume concentration of 90% and 4 column volumes of ethanol with a volume concentration of 60%. The ethanol eluate with a volume concentration of 60% is collected, ethanol is recovered under reduced pressure, and dried to obtain the crude product.

13. The preparation method of genipin-1-β-D-gentiobioside according to claim 10, characterized in that: In step c, the method of hot dissolution and recrystallization of the crude product with ethanol is specifically as follows: The crude product is added with 0.5 - 1 times anhydrous ethanol, heated under reflux to dissolve it, filtered while hot, left to stand for precipitation, and filtered by suction to obtain the refined product.

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